Methods and compositions for the clinical derivation of a stem cell and therapeutic uses

ABSTRACT

Various cells, stem cells, and stem cell components, including associated methods of generating and using such cells are provided. In one aspect, for example, an isolated cell that is capable of self-renewal and culture expansion and is obtained from a subepithelial layer of a mammalian umbilical cord tissue. Such an isolated cell expresses at least three cell markers selected from CD29, CD73, CD90, CD166, SSEA4, CD9, CD44, CD146, or CD105, and does not express at least three cell markers selected from CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19, CD117, Stro-1, or HLA-DR.

PRIORITY DATA

This application is a continuation of U.S. patent application Ser. No.17/322,672, filed on May 17, 2021, which is a continuation of U.S.patent application Ser. No. 15/799,743, filed on Oct. 31, 2017, which iscontinuation of U.S. patent application Ser. No. 13/732,204, filed onDec. 31, 2012, now issued as U.S. Pat. No. 9,803,176, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 61/582,070,filed on Dec. 30, 2011, and of U.S. Provisional Patent Application Ser.No. 61/591,211, filed on Jan. 26, 2012, each of which are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to stem cells and variousrelated aspects thereof. Accordingly, the present invention involves thefields of chemistry, life science, and medicine.

BACKGROUND

Various cell and stem cell populations have been shown to have value forresearch applications. However, clinical translation of these cell typesfor human and animal use in therapeutic applications is limited due to anumber of reasons, including allogenic issues.

SUMMARY

The present disclosure provides various cells, stem cells, and stem cellcomponents, including associated methods of generating and using suchcells. In one aspect, for example, an isolated cell that is capable ofself-renewal and culture expansion and is obtained from a subepitheliallayer of a mammalian umbilical cord tissue is provided. Such an isolatedcell expresses at least three cell markers selected from CD29, CD73,CD90, CD166, SSEA4, CD9, CD44, CD146, or CD105, and does not express atleast three cell markers selected from CD45, CD34, CD14, CD79, CD106,CD86, CD80, CD19, CD117, Stro-1, or HLA-DR. In another aspect, theisolated cell expresses CD29, CD73, CD90, CD166, SSEA4, CD9, CD44,CD146, and CD105. In yet another aspect, the isolated cell does notexpress CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19, CD117, Stro-1,and HLA-DR. In some aspects, the isolated cell can be positive for SOX2,OCT4, or both SOX2 and OCT4. In a further aspect, the isolated cell canproduce exosomes expressing CD63, CD9 or both. It is understood that thepresent scope includes cultures of isolated cells.

The cells according to aspects of the present disclosure are capable ofdifferentiation into a variety of cell types, and any such cell type isconsidered to be within the present scope. Non-limiting examples of suchcell types can include adipocytes, chondrocytes, osteocytes,cardiomyocytes, endothelial cells, myocytes, and the like, includingcombinations thereof.

A variety of cells and cellular products can be derived from theisolated cells described herein, and any such cells and cellularproducts are considered to be within the present scope. In one aspect,for example, the present disclosure provides an isolated exosome derivedfrom the isolated cells described, where the exosome expresses CD63, CD9or both. In another aspect, an adipocyte cell that has beendifferentiated from the isolated cells described is provided. In yetanother aspect, a chondrocyte cell that has been differentiated from theisolated cells described is provided. In a further aspect, an osteocytecell that has been differentiated from the isolated cells described isprovided. In yet a further aspect, a cardiomyocyte cell that has beendifferentiated from the isolated cells described is provided.Furthermore, a culture of differentiated cells derived from the isolatedcells described including at least one cell type selected from anadipocyte, a chondrocyte, an osteocyte, or a cardiomyocyte is provided.

In another aspect, the present disclosure provides a method of culturingstem cells from a subepithelial layer of a mammalian umbilical cord.Such a method can include dissecting the subepithelial layer from theumbilical cord, placing the dissected subepithelial layer interior sidedown on a substrate such that an interior side of the subepitheliallayer is in contact with the substrate, and culturing the subepitheliallayer on the substrate. The method can additionally include removingexplants for primary cell expansion. In one aspect, dissecting thesubepithelial layer further includes removing Wharton's Jelly from theumbilical cord.

The subepithelial layer can be cultured in any media capable ofproducing explants therefrom, and any such medium is considered to bewithin the present scope. In one specific aspect, however, one suchculture medium can include a platelet lysate. In another aspect, theculture media can include human or animal platelet lysate. In yetanother aspect, the culture media can be derived from human-free andanimal-free ingredients.

The substrate utilized to culture the subepithelial layer can be anysubstrate capable of deriving explants therefrom. In one aspect, thesubstrate can be a polymeric matrix. One example of such a polymericmatrix is a culture dish. In one specific aspect, the culture dish canbe a cell culture treated plastic, and the subepithelial layer can beplaced thereon without any additional pretreatment to the cell culturetreated plastic. In another aspect, the substrate can be a semi-solidcell culture substrate. Any type of semi-solid substrate that is capableof supporting the subepithelial layer during the culturing procedure isconsidered to be within the present scope.

Various culturing conditions are contemplated, and it is understood thatsuch conditions can vary depending on experimental protocol and variousdesired results. In one aspect, for example, the subepithelial layer canbe cultured in a normoxic environment. In another aspect, thesubepithelial layer can be cultured in a hypoxic environment.Additionally, in some aspects, the culturing of the subepithelial layerand the removal of the explants can be performed without the use of anyenzymes. Furthermore, in some aspects, subculturing of the explantsand/or the cells resulting from the explants can be performed withoutthe use of any enzymes.

In yet another aspect of the present disclosure, a method of treating amedical condition responsive to treatment with the isolated cellsdescribed herein can include introducing such cells into an individualhaving the medical condition. These cellular treatments can be utilizedto treat any condition for which they are capable providing a benefit.Non-limiting examples of such medical conditions include COPD, diabetes,ischemia, osteoarthritis, orthopedic damage, liver damage, chronicrefractory angina, erectile dysfunction, herniated disks, congestiveheart failure, asthma, emphysema, wounds, acute radiation syndrome,autoimmune disorders, ischemic organ beds, graft vs. host disease, andthe like, including combinations thereof. Additionally, in anotheraspect, a method of treating a medical condition responsive to treatmentwith the differentiated cells described herein can include introducingat least one cell type of the differentiated cells into an individualhaving the medical condition.

In a further aspect, a method of treating COPD is provided. Such amethod can include administering a COPD effective active agentintravenously to a subject to deliver the COPD effective active agent toa lower half of the subject's lung, and administering the COPD effectiveactive agent in an aerosolized form to the subject via ventilation todeliver the COPD effective active agent to an upper half of thesubject's lung. In one aspect, the COPD effective active agent includesstem cells. In yet another aspect, the stem cells include cells derivedfrom the subepithelial layer of a mammalian umbilical cord as has beendescribed herein. In one specific aspect, the stem cells can beaerosolized with an aerosolizer to a size of from about 6 to about 200microns. Additionally, the two types of administration can be deliveredsequentially or concomitantly.

In another aspect, the COPD effective active agent can be an activeagent other than stem cells. Non-liming examples of such COPD effectiveactive agents can include exosomes, cell lysates, protein extractsderived from cell culture, and the like, including combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an image of a histological section of umbilical cordidentifying the subepethelial layer in accordance with one aspect of thepresent disclosure.

FIG. 2A shows explant of cells migrating from the subepithelial layerand karyotyping of cells in accordance with another aspect of thepresent disclosure.

FIG. 2B shows explant of cells migrating from the subepithelial layerand karyotyping of cells in accordance with another aspect of thepresent disclosure.

FIG. 2C shows karyotyping of cells in accordance with another aspect ofthe present disclosure

FIG. 3A shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3B shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3C shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3D shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3E shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3F shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3G shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3H shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3I shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3J shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3K shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3L shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3M shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3N shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 3O shows FACS analysis of cell determinant markers expressed bycells or stem cells derived from umbilical cord in accordance withanother aspect of the present disclosure.

FIG. 4A shows images of RT-PCR analysis of RNA extracted from cells orstem cells derived from umbilical cord in accordance with another aspectof the present disclosure.

FIG. 4B shows images of immunocytochemical staining of cells inaccordance with another aspect of the present disclosure.

FIG. 4C shows images of immunocytochemical staining of cells inaccordance with another aspect of the present disclosure.

FIG. 4D shows images of immunocytochemical staining of cells inaccordance with another aspect of the present disclosure.

FIG. 5A shows images of culture of cells or stem cells derived fromumbilical cord tissue in semi-solid PRP matrix or PL Lysate inaccordance with another aspect of the present disclosure.

FIG. 5B shows images of culture of cells or stem cells derived fromumbilical cord tissue in semi-solid PRP matrix or PL Lysate inaccordance with another aspect of the present disclosure.

FIG. 6A shows extracellular exosome size analysis in accordance withanother aspect of the present disclosure.

FIG. 6B shows an SEM of exosomes in accordance with another aspect ofthe present disclosure.

FIG. 6C shows CD63 expression of exosomes produced from cells or stemcells derived from umbilical cord in accordance with another aspect ofthe present disclosure.

FIG. 6D shows CD63 expression of exosomes produced from cells or stemcells derived from umbilical cord in accordance with another aspect ofthe present disclosure.

FIG. 7A shows images demonstrating differentiation of umbilical cordtissue into adipogenic lineages in accordance with another aspect of thepresent disclosure.

FIG. 7B shows images demonstrating differentiation of umbilical cordtissue into adipogenic lineages in accordance with another aspect of thepresent disclosure.

FIG. 7C shows images demonstrating differentiation of umbilical cordtissue into adipogenic lineages in accordance with another aspect of thepresent disclosure.

FIG. 7D shows images demonstrating differentiation of umbilical cordtissue into adipogenic lineages in accordance with another aspect of thepresent disclosure.

FIG. 8A shows images demonstrating differentiation of umbilical cordtissue into osteogenic lineages in accordance with another aspect of thepresent disclosure.

FIG. 8B shows images demonstrating differentiation of umbilical cordtissue into osteogenic lineages in accordance with another aspect of thepresent disclosure.

FIG. 8C shows images demonstrating differentiation of umbilical cordtissue into osteogenic lineages in accordance with another aspect of thepresent disclosure.

FIG. 8D shows images demonstrating differentiation of umbilical cordtissue into osteogenic lineages in accordance with another aspect of thepresent disclosure.

FIG. 9A shows an image demonstrating differentiation of umbilical cordtissue into Chondrogenic lineages in accordance with another aspect ofthe present disclosure.

FIG. 9B shows an image demonstrating differentiation of umbilical cordtissue into Chondrogenic lineages in accordance with another aspect ofthe present disclosure.

FIG. 10A shows an image demonstrating differentiation of umbilical cordtissue into cardiogenic lineages in accordance with another aspect ofthe present disclosure.

FIG. 10B shows an image demonstrating differentiation of umbilical cordtissue into cardiogenic lineages in accordance with another aspect ofthe present disclosure.

FIG. 10C shows an image demonstrating differentiation of umbilical cordtissue into cardiogenic lineages in accordance with another aspect ofthe present disclosure.

FIG. 10D shows an image demonstrating differentiation of umbilical cordtissue into cardiogenic lineages in accordance with another aspect ofthe present disclosure.

FIG. 11A shows data relating to chronic limb ischemia and painperception over time in accordance with another aspect of the presentdisclosure.

FIG. 11B shows data relating to chronic limb ischemia and painperception over time in accordance with another aspect of the presentdisclosure.

FIG. 12 shows an image of an angiogram demonstrating delivery of cellsinto the heart in accordance with another aspect of the presentdisclosure.

FIG. 13A shows an image in a series of images of an angiogramdemonstrating delivery of cells into the heart in accordance withanother aspect of the present disclosure.

FIG. 13B shows an image in a series of images of an angiogramdemonstrating delivery of cells into the heart in accordance withanother aspect of the present disclosure.

FIG. 13C shows an image in a series of images of an angiogramdemonstrating delivery of cells into the heart in accordance withanother aspect of the present disclosure.

FIG. 13D shows an image in a series of images of an angiogramdemonstrating delivery of cells into the heart in accordance withanother aspect of the present disclosure.

FIG. 14A shows an image of a series of images of the knee of an 80 yearold female prior to and following the delivery of stem cells into theintraarticular space in accordance with another aspect of the presentdisclosure.

FIG. 14B shows an image of a series of images of the knee of an 80 yearold female prior to and following the delivery of stem cells into theintraarticular space in accordance with another aspect of the presentdisclosure.

FIG. 14C shows an image of a series of images of the knee of an 80 yearold female prior to and following the delivery of stem cells into theintraarticular space in accordance with another aspect of the presentdisclosure.

FIG. 14D shows an image of a series of images of the knee of an 80 yearold female prior to and following the delivery of stem cells into theintraarticular space in accordance with another aspect of the presentdisclosure.

FIG. 15A shows data relating to acute radiation syndrome in accordancewith another aspect of the present disclosure.

FIG. 15B shows data relating to acute radiation syndrome in accordancewith another aspect of the present disclosure.

FIG. 16 shows data relating to acute radiation syndrome in accordancewith another aspect of the present disclosure.

DETAILED DESCRIPTION

Before the present disclosure is described herein, it is to beunderstood that this disclosure is not limited to the particularstructures, process steps, or materials disclosed herein, but isextended to equivalents thereof as would be recognized by thoseordinarily skilled in the relevant arts. It should also be understoodthat terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting.

Definitions

The following terminology will be used in accordance with thedefinitions set forth below.

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” and, “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a cell” includes one or more of such cells and referenceto “the flask” includes reference to one or more of such flasks.

As used herein, the term “isolated cell” refers to a cell that has beenisolated from the subepithelial layer of a mammalian umbilical cord.

As used herein, the term “substantially” refers to the complete ornearly complete extent or degree of an action, characteristic, property,state, structure, item, or result. For example, an object that is“substantially” enclosed would mean that the object is either completelyenclosed or nearly completely enclosed. The exact allowable degree ofdeviation from absolute completeness may in some cases depend on thespecific context. However, generally speaking the nearness of completionwill be so as to have the same overall result as if absolute and totalcompletion were obtained. The use of “substantially” is equallyapplicable when used in a negative connotation to refer to the completeor near complete lack of an action, characteristic, property, state,structure, item, or result. For example, a composition that is“substantially free of” particles would either completely lackparticles, or so nearly completely lack particles that the effect wouldbe the same as if it completely lacked particles. In other words, acomposition that is “substantially free of” an ingredient or element maystill actually contain such item as long as there is no measurableeffect thereof.

As used herein, the term “about” is used to provide flexibility to anumerical range endpoint by providing that a given value may be “alittle above” or “a little below” the endpoint.

As used herein, a plurality of items, structural elements, compositionalelements, and/or materials may be presented in a common list forconvenience. However, these lists should be construed as though eachmember of the list is individually identified as a separate and uniquemember. Thus, no individual member of such list should be construed as ade facto equivalent of any other member of the same list solely based ontheir presentation in a common group without indications to thecontrary.

Concentrations, amounts, and other numerical data may be expressed orpresented herein in a range format. It is to be understood that such arange format is used merely for convenience and brevity and thus shouldbe interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range, but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited. Asan illustration, a numerical range of “about 1 to about 5” should beinterpreted to include not only the explicitly recited values of about 1to about 5, but also include individual values and sub-ranges within theindicated range. Thus, included in this numerical range are individualvalues such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4,and from 3-5, etc., as well as 1, 2, 3, 4, and 5, individually.

This same principle applies to ranges reciting only one numerical valueas a minimum or a maximum. Furthermore, such an interpretation shouldapply regardless of the breadth of the range or the characteristicsbeing described.

THE DISCLOSURE

The present disclosure presents a novel discovery of an allogenic cellor stem cell population that can be used for treating a wide range ofconditions. In addition this disclosure describes a novel media andmethod of culturing these cells without, in some cases, the use ofanimal products or enzymes. As such, cells, stem cells, cell cultures,and associated methods, including methods of isolating, culturing,developing, or otherwise producing these cells are provided. The scopeof the present disclosure additionally encompasses research andtherapeutic uses of such cell and cell cultures, including compoundsderived therefrom.

As one example, the cell and stem cell populations and compounds derivedfrom these populations may be used in allogenic applications to treat awide range of conditions including, but not limited to, cardiac,orthopedic, autoimmune, diabetes, cardio vascular disorders,neurological, erectile dysfunction, spinal cord injuries, herniateddisks, critical limb ischemia, hypertension, wound healing, ulcers,chronic obstructive lung disease, acute radiation syndrome, graft vs.host disease, ischemic organ beds and the like. Also described aremethods of producing cell and stem cell populations and compounds thatmay be used for drug discovery and development, as well as toxicologytesting. Examples of compounds derived from these cell and stem cellpopulations are small vesicles that contain proteins, RNA, micro RNAs,and the like, that are specific to the cell and stem cell populations.

In one aspect, an isolated cell obtained from a subepithelial layer of amammalian umbilical cord tissue capable of self-renewal and cultureexpansion is provided. Such a cell is capable of differentiation into acell type such as, in one aspect for example, adipocytes, chondrocytes,osteocytes, cardiomyocytes, and the like. In another aspect,non-limiting examples of such cell types can include white, brown, orbeige adipocytes, chondrocytes, osteocytes, cardiomyocytes, endothelialcells, myocytes, and the like, including combinations thereof. Otherexamples of such cell types can include neural progenitor cells,hepatocytes, islet cells, renal progenitor cells, and the like.

A cross section of a human umbilical cord is shown in FIG. 1, whichshows the umbilical artery (UA), the umbilical veins (UV), the Wharton'sJelly (WJ), and the subepithelial layer (SL). Isolated cells from the SLcan have a variety of characteristic markers that distinguish them fromcell previously isolated from umbilical cord samples. It should be notedthat these isolated cells are not derived from the Wharton's Jelly, butrather from the SL.

Various cellular markers that are either present or absent can beutilized in the identification of these SL-derived cells, and as such,can be used to show the novelty of the isolated cells. For example, inone aspect, the isolated cell expresses at least three cell markersselected from CD29, CD73, CD90, CD146, CD166, SSEA4, CD9, CD44, CD146,or CD105, and the isolated cell does not express at least three cellmarkers selected from CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19,CD117, Stro-1, or HLA-DR. In another aspect, the isolated cell expressesat least five cell markers selected from CD29, CD73, CD90, CD146, CD166,SSEA4, CD9, CD44, CD146, or CD105. In another aspect, the isolated cellexpresses at least eight cell markers selected from CD29, CD73, CD90,CD146, CD166, SSEA4, CD9, CD44, CD146, or CD105. In a yet anotheraspect, the isolated cell expresses at least CD29, CD73, CD90, CD166,SSEA4, CD9, CD44, CD146, and CD105. In another aspect, the isolated celldoes not express at least five cell markers selected from CD45, CD34,CD14, CD79, CD106, CD86, CD80, CD19, CD117, Stro-1, or HLA-DR. Inanother aspect, the isolated cell does not express at least eight cellmarkers selected from CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19,CD117, Stro-1, or HLA-DR. In yet another aspect, the isolated cell doesnot express at least CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19,CD117, Stro-1, and HLA-DR. Additionally, in some aspects, the isolatedcell can be positive for SOX2, OCT4, or both SOX2 and OCT4. In a furtheraspect, the isolated cell can produce exosomes expressing CD63, CD9, orboth CD63 and CD9.

A variety of techniques can be utilized to extract the isolated cells ofthe present disclosure from the SL, and any such technique that allowssuch extraction without significant damage to the cells is considered tobe within the present scope. In one aspect, for example, a method ofculturing stem cells from the SL of a mammalian umbilical cord caninclude dissecting the subepithelial layer from the umbilical cord. Inone aspect, for example, umbilical cord tissue can be collected andwashed to remove blood, Wharton's Jelly, and any other materialassociated with the SL. For example, in one non-limiting aspect the cordtissue can be washed multiple times in a solution of Phosphate-BufferedSaline (PBS) such as Dulbecco's Phosphate-Buffered Saline (DPBS). Insome aspects the PBS can include a platelet lysate (i.e. 10% PRP lysateof platelet lysate). Any remaining Wharton's Jelly or gelatinous portionof the umbilical cord can then be removed and discarded. The remainingumbilical cord tissue (the SL) can then be placed interior side down ona substrate such that an interior side of the SL is in contact with thesubstrate. An entire dissected umbilical cord with the Wharton's Jellyremoved can be placed directly onto the substrate, or the dissectedumbilical cord can be cut into smaller sections (e.g. 1-3 mm) and thesesections can be placed directly onto the substrate.

A variety of substrates are contemplated upon which the SL can beplaced. In one aspect, for example, the substrate can be a solidpolymeric material. One example of a solid polymeric material caninclude a cell culture dish. The cell culture dish can be made of a cellculture treated plastic as is known in the art. In one specific aspect,the SL can be placed upon the substrate of the cell culture dish withoutany additional pretreatment to the cell culture treated plastic. Inanother aspect, the substrate can be a semi-solid cell culturesubstrate. Such a substrate can include, for example, a semi-solidculture medium including an agar or other gelatinous base material.

Following placement of the SL on the substrate, the SL is cultured in asuitable medium. In some aspects it is preferable to utilized culturemedia that is free of animal and human components or contaminants. Asone example, FIG. 2 shows the culturing of cells from the SL. As can beseen in FIG. 2A, at three days post plating of the SL, cells have begunto migrate. FIG. 2B shows cells after 6 days of culture in animal freemedia. Furthermore, FIG. 2C shows the karyotype of cells followingpassage 12. As has been described, the cells derived from the SL have aunique marker expression profile. Data showing a portion of this profileis shown in FIGS. 3A-O.

The culture can then be cultured under either normoxic or hypoxicculture conditions for a period of time sufficient to establish primarycell cultures. (e.g. 3-7 days in some cases). After primary cellcultures have been established, the SL tissue is removed and discarded.Cells or stem cells are further cultured and expanded in larger cultureflasks in either a normoxic or hypoxic culture conditions. While avariety of suitable cell culture media are contemplated, in onenon-limiting example the media can be Dulbecco's Modified Eagle Medium(DMEM) glucose (500-6000 mg/mL) without phenol red, 1× glutamine,1×NEAA, and 0.1-20% PRP lysate or platelet lysate. Another example ofsuitable media can include a base medium of DMEM low glucose withoutphenol red, 1× glutamine, 1×NEAA, 1000 units of heparin and 20% PRPlysate or platelet lysate. In another example, cells can be cultureddirectly onto a semi-solid substrate of DMEM low glucose without phenolred, 1× glutamine, 1×NEAA, and 20% PRP lysate or platelet lysate. In afurther example, culture media can include a low glucose medium(500-1000 mg/mL) containing 1× Glutamine, 1×NEAA, 1000 units of heparin.In some aspects, the glucose can be 1000-4000 mg/mL, and in otheraspects the glucose can be high glucose at 4000-6000 mg/mL. These mediacan also include 0.1%-20% PRP lysate or platelet lysate. In yet afurther example, the culture medium can be a semi-solid with thesubstitution of acid-citrate-dextrose ACD in place of heparin, andcontaining low glucose medium (500-1000 mg/mL), intermediate glucosemedium (1000-4000 mg/mL) or high glucose medium (4000-6000 mg/mL), andfurther containing 1× Glutamine, 1×NEAA, and 0.1%-20% PRP lysate orplatelet lysate. In some aspects, the cells can be derived, subcultured,and/or passaged using TrypLE. In another aspect, the cells can bederived, subcultured, and/or passaged without the use of TrypLE or anyother enzyme.

FIG. 4 shows data relating to various genetic characteristics of thecells isolated from the SL tissue. FIG. 4A shows that isolated SL cells(lane 1) are positive for SOX2 and OCT4, and are negative for NANOG ascompared to control cells (Ctrl). FIG. 4B shows a DAPI stained image ofcultured SL cells demonstrating that such cells are positive for CD44.FIG. 4C shows a DAPI stained image of cultured SL cells demonstratingthat such cells are positive for CD90. FIG. 4D shows a DAPI stainedimage of cultured SL cells demonstrating that such cells are positivefor CD146.

In one aspect, SL cells can be cultured from a mammalian umbilical cordin a semi-solid PRP Lysate or platelet lysate substrate. Such cells canbe cultured directly onto a plastic coated tissue culture flask as hasbeen described elsewhere herein. After a sufficient time in eithernormoxic or hypoxic culture environments the media is changed andfreshly made semi-solid PRP lysate or platelet lysate media is added tothe culture flask. The flask is continued to be cultured in either anormoxic or hypoxic culture environment. The following day the mediabecomes a semi-solid PRP-lysate or platelet lysate matrix. The cells canbe continued to be cultured in this matrix being until further use.FIGS. 5A and B show SL cells growing in a semi-solid PRPL or PL gel at10× and 40× magnifications. In one specific aspect, ingredients for asemi solid culture can include growth factors for expanded cell cultureof differentiation. Non-limiting examples can include FGF, VEGF, FNDC5,5-azacytidine, TGF-Beta1, TGF Beta2, insulin, ITS, IGF, and the like,including combinations thereof.

In some cases, allogenic confirmation of SL cells, either differentiatedor undifferentiated, can be highly beneficial, particularly fortherapeutic uses of the cells. In such cases, mixed lymphocyte reactionscan be performed on the cells to confirm the allogenic properties of thecells. In certain aspects, a cell derived as described herein does notcause a mixed lymphocyte response or T-cell proliferation.

In certain aspects, a cell derived as described herein can berecombinantly modified to express one or more genes and or proteins. Inone technique, a gene or genes can be incorporated into an expressionvector. Approaches to deliver a gene into the cell can include withoutlimitation, viral vectors, including recombinant retroviruses,adenoviruses, adeno-associated virus, lentivirus, poxivirus, alphavirus,herpes simplex virus-1, recombinant bacterial, eukaryotic plasmids, andthe like, including combinations thereof. Plasmid DNA may be deliverednaked or with the help of exosomes, cationic liposomes or derivatized(antibody conjugated) polylysine conjugates, gramicidin S, artificialviral envelopes, other intracellular carriers, as well as directinjection of the genes. In some aspects, non-viral gene delivery methodscan be used, such as for example, scaffold/matrix attached region(S/MAR)-based vector.

Furthermore, in some aspects, isolated SL cells can be used to producean exosome population. These exosome populations can be utilized for avariety of research and therapeutic uses. In one aspect, for example,cells are cultured as described in either a normoxic or hypoxic cultureenvironment and supernatants are collected at each media change.Exosomes can then be purified from the supernatants using an appropriatepurification protocol. One not-limiting example of such a protocol isthe ExoQuick isolation system by SYSTEMBIO. Purified exosomes can beutilized for further manipulation, targeting, and therapeutic use. Theexosomes specific to the SL cells are positive for CD63 expression. FIG.6A shows an analysis of the size of exosomes obtained as has beendescribed, and FIG. 6B shows and electron microscope image of a samplingof exosomes. Additionally, FIGS. 6C-D show CD63 expression of exosomesproduced from cells or stem cells derived from umbilical cord.

In some aspects, the isolated cells and cell cultures can be utilizedas-is upon isolation from the SL tissue. In other aspects, the isolatedcells can be differentiated into other cell types. It should be notedthat any useful cell type that can be derived from the cells isolatedfrom SL tissue are considered to be within the present scope.Non-limiting examples of such cell types include adipocytes,chondrocytes, osteocytes, cardiomyocytes, and the like. Differentiationcan be induced by exposing the cells to chemicals, growth factors,supernatants, synthetic or naturally occurring compounds, or any otheragent capable of transforming the cells. In one aspect, for example, theisolated cells can be differentiated into adipocytes, as is shown inFIG. 7.

Any technique for differentiation of SL cells into adipocytes isconsidered to be within the present scope. One non-limiting example usedfor adipogenic differentiation includes SL cells cultured in thepresence of StemPro Adipogenic Differentiation media (LifeTechnologies). FIG. 7A shows differentiated SL cells that are positivefor the adipogenic markers FABP4, LPL, and PPARy (lane 1). Foradipogenic differentiation, confirmation was determined by Oil Red Ostaining and FABP4 immunocytochemistry. FIG. 7B shows an image of DAPIstained cells showing FABP4 markers. FIG. 7C shows unstained cells andFIG. 7D shows Oil Red O staining demonstrating the storage of fats inthe cells.

For osteogenic differentiation of SL cells, one non-limiting techniquecultures such cells in the presence of StemPro OsteogenicDifferentiation media (Life Technologies). As is shown in FIG. 8A, forexample, differentiated SL cells are positive for the osteogenic markersOP, ON, and AP (lane 1). For osteogenic differentiation, confirmationwas determined by Alizarin red staining and osteocalcinimmunocytochemistry. FIG. 8B shows an image of DAPI stained cellsshowing the presence of osteocalcin. FIG. 8C shows unstained cells andFIG. 8D shows an image of cells stained with alizarin red demonstratingthe presence of calcific deposition in the cells.

For chondrogenic differentiation of SL cells, one non-limiting techniquecultures SL cells in the presence of StemPro ChondrogenicDifferentiations media (Life Technologies). As is shown in FIG. 9A,differentiated SL cells are positive for chondrogenic markers Collagen2A, A6, and BG (lane 1). For chondrogenic differentiation, confirmationwas determined by Von Kossa staining. FIG. 9B shows Alcian blue stainingof a chondrocyte pellet.

For cardiogenic differentiation of SL cells, one non-limiting techniquecultures cells in the presence of DMEM low glucose without phenol red,1× glutamine, 1×NEAA and 10% PRP lysate or platelet lysate with 5-10 μM5-AZA-2′-deoxycytidine. As is shown in FIG. 10A, differentiated SL cellsare positive for the cardiogenic markers MYF5, CNX43, and ACTIN (lane1). For cardiogenic differentiation, confirmation was determined bystaining for ANP, tropomyosin, and troponin 1. FIG. 10B shows an imageof DAPI stained cells demonstrating the presence of Troponin 1. FIG. 10Cshows an image of DAPI stained cells demonstrating the presence oftropomyosin. FIG. 10D shows a merged image of the images from FIGS. 10Band 10C.

In yet another aspect, a method of treating a medical condition isprovided. In some embodiments, such a method can include introducingcells described herein into an individual having the medical condition.Cells can be delivered at various doses such as, without limitation,from about 500,000 to about 1,000,000,000 cells per dose. In someaspects, the cell dosage range can be calculated based on the subject'sweight. In certain aspects, the cell range is calculated based on thetherapeutic use or target tissue or method of delivery. Non-limitingexamples of medical conditions can include COPD, diabetes, ischemia,osteoarthritis, orthopedic damage, liver damage, chronic refractoryangina, congestive heart failure, asthma, emphysema, wounds, erectiledysfunction, spinal cord injuries, herniated disks, acute radiationsyndrome, neurological disorders, graft vs. host disease, autoimmunedisorders, renal failure, autoimmune disorders, and the like, includingcombinations thereof. The treatment can include introducing cells into aregion of the subject where the medical condition can be treated. Thecells can be delivered intramuscularly, intravenously, intraarterially,subcutaneously, surgically, intrathecally, intraperitoneally,intranasally, orally, topically, rectally, vaginally, via aspiration,and the like, including combinations thereof. Additionally, in oneaspect, undifferentiated SL cells can be delivered to the subject totreat the medical condition. In another aspect, differentiated SL cellscan be delivered to the subject to treat the medical condition.

Stem cells can also be delivered into an individual according toretrograde or antegrade delivery. As an example, cells can be introducedinto an organ of an individual via retrograde delivery of the cells intothe organ. Non-limiting examples of such organs can include the heart,the liver, a kidney, the brain, pancreas, and the like.

Additionally, in some aspects SL cells can be lysed and the lysate usedfor treatment. In other aspects, supernatant from the culture processcan be used for treatment. One example of such a supernatant treatmentincludes the delivery of exosomes. Exosomes can be delivered into theindividual via aerosolized delivery, IV delivery, or any other effectivedelivery technique. Exosomes can also be used to treat individuals withopen wounds, ulcers, burns, and the like.

In a further aspect, a method of treating COPD is provided. Such amethod can include administering a COPD effective active agentintravenously to a patient to deliver the COPD effective active agent toa lower half of the patient's lung, and also administering the COPDeffective active agent in an aerosolized form to the patient viaventilation to deliver the COPD effective active agent to an upper halfof the patient's lung. In some embodiments, the administration can beconcomitant. In other aspects, the administration can be sequential. Insome aspects, the COPD effective agent delivered intravenously can bedifferent from the COPD effective agent delivered in aerosol form, whilein other aspects the same COPD effective agent can be utilized in bothadministrations. In some cases it can be beneficial for the patient tobe in a sitting position during delivery of the COPD effective activeagent. In one aspect, the COPD effective active agent includes stemcells. In another aspect, the stem cells include the cells describedherein. In another aspect, the active agent can be a pharmaceuticalagent, or a biologic agent. Other non-limiting examples of COPDeffective active agents can include exosomes, cell lysates, proteinextracts, protein extracts derived from cell culture, and the like.

A variety of conditions can be utilized to aerosolize cells. In oneaspect, for example, cells can be suspended in 1-5 mls of saline andaerosolized at a pressure of 3-100 psi for 1-15 minutes, or until thecells begin to rupture and/or die.

Any form of aerosolizer can be utilized to deliver stem cells to thelungs provided the stem cells can be delivered substantially withoutdamage. In some cases, it can be beneficial to aerosol stem cells via anaerosolizer capable of aerosolizing particles to larger sizes. Forexample, in one aspect, an aerosolizer can be used that aerosolizes to aparticle size of from about 2 microns to about 50 microns. In anotheraspect, an aerosolizer can be used that aerosolizes to a particle sizeof from about 4 microns to about 30 microns. In yet another aspect, anaerosolizer can be used that aerosolizes to a particle size of fromabout 6 microns to about 20 microns. In yet another aspect, anaerosolizer can be used that aerosolizes to a particle size of fromabout 6 microns to about 200 microns.

In another example, the present techniques can be utilized in thetreatment of acute radiation syndrome. Acute radiation syndrome can bechallenging to treat, with survival being dependent on the dose ofradiation and the subsequent clinical care to mediate lethal infections,including providing support for resident stem cell expansion.Traditional techniques utilize growth factor treatment or hematopoeiticstem cell transplantation. The stem cells according to aspects of thepresent disclosure can be used under allogeneic transplant models withno HLA matching needed between donor and host. The cells have been shownto be hypoimmunogenic and not recognized by the immune system, evenfollowing multiple injections. These stem cells secrete severalbioactive molecules, such as hematopoietic growth factors including IL6,IL11, LIF SCF and Fly3 ligand and immunomodulatory molecules such asTGFB1, prostaglandin E2, indoleamine 2,3-dioxygenase.

Such cultured cells facilitate a protective mechanism combating theinflammatory cascade in addition to supporting detoxification afterradiation exposure. In addition, these cells release trophic factors andHSC-niche modulating activity to rescue endogenous hematopoiesis andactivity. This data suggest that these cells serve as a fast andeffective treatment in a first-line of defense to combatradiation-induced hematopoietic failure. In addition these cells may beused to treat severe or steroid resistant graft vs. host disease.

EXAMPLES Example 1—Composition for Culturing Cells or Stem Cell fromUmbilical Cord for Clinical Use Media Composition-1 DMEM-LowGlucose—Phenol Free 1× Glutamine 1×NEAA

10% PRP Lysate or platelet lysate1000 units of heparin

Media Composition-2 DMEM-Low Glucose—Phenol Free 1× Glutamine 1×NEAA

Lyophilized 10% PRP Lysate or platelet lysate Tablet1000 units of heparin

Media Composition-3 DMEM-Low Glucose—Phenol Free 1× Glutamine 1×NEAA

10% PRP Lysate or platelet lysate

ACD Example 2—Culturing Cells or Stem Cell from Umbilical Cord forClinical Use

Umbilical cord tissue is obtained and maternal blood is tested forinfectious disease prior to derivation of cell and stem cellpopulations. A 1 cm piece of cord is washed 10 times in a solution ofDPBS containing 10% PRP-Lysate or platelet lysate. The umbilical cord isthen opened longitudinally to expose the interior of the umbilical cord.All tissue is removed that can give rise to endothelial cells. Theumbilical cord is then place directly into a cell culture dishcontaining Media Composition-1 with the interior of the umbilical cordin contact with the plastic and cultured in either normoxic or hypoxicculture environments.

On the third day the media is replaced with fresh Media Composition-1and cultured until day seven when the explants are removed for primarycell expansion. The cells are fed every other day until approximately500,000-1,000,000 cells can be harvested and further expanded. It isnoted that the media used for subsequent examples is Media Composition-1unless specifically noted otherwise.

Example 3—Enzymatic Passage of Cells or Stem Cell from Umbilical Cordfor Clinical Use

TrypLE can be used for subculturing the cells. The media is removed fromthe flask of Example 2 and the cells are washed three times with DPBS.TrypLE is then added and the cells are transferred to the incubator at37 C for 3-5 minutes. The enzymatic reaction is stopped by the additionof equal volume of culture/expansion media. The cells are thencentrifuged 400×g for 5 minutes at room temperature. The supernatant isremoved and the cells are washed 3 times if they will be furthersubcultured or 10 times if they will be used therapeutically.

Example 4—Non-Enzymatic Passage of Cells or Stem Cell from UmbilicalCord for Clinical Use

For a non-enzymatic approach, a semi-solid gel can be used to remove thecells from the tissue culture flask. The cells are cultured in normalculture/expansion media. One day prior to subculture, freshly preparedDMEM-Low Glucose—Phenol Free, 1× Glutamine, 1×NEAA, 10% PRP Lysate orplatelet lysate, ACD semi-solid media is added to the cells. The cellsare cultured overnight under either a normoxic or hypoxic environment.The following day a semi-solid gel is formed over the cells. To removethe cells from the dish, the side of the dish is tapped until thesemi-solid gel is dislodged from the bottom. This semi-solid layer canthen be removed, and the cells will be located within the semi-solidgel. If further subculture is required the semi-solid gel is transferredto additional cell culture flasks or bags for further expansion. If thecells are not to be further expanded the semi-solid layer containing thecells can be directly applied therapeutically.

Example 5—Therapeutic Use of Cells or Stem Cells from Umbilical Cord forTreating Critical Limb Ischemia

Patients qualified for inclusion if they had chronic, critical limbischemia including rest pain (Rutherford class 4) or mild-to-moderatetissue loss (Rutherford 5) and were not candidates for surgical orendovascular revascularization. Hemodynamic parameters included one ofthe following: ankle pressure <50 mmHg or ABI <0.4; toe pressure <40mmHg or TBI <0.4; or TcPO2 <20 mmHg on room air.

Exclusion criteria included extensive necrosis of the index limb makingamputation inevitable (Rutherford class 6); uncorrected iliac arteryocclusion ipsilateral to index limb; lack of Doppler signal in the indexlimb (ABI=0); serum creatinine ≥2.0 mg/dL; active infection requiringantibiotics; active malignancy; or any hematologic disorder thatprevented bone marrow harvesting.

All patients were ≥18 years of age and able to provide informed consent.All enrolled patients underwent pre-operative cancer screening andophthalmologic examinations for proliferative retinopathy.

Cells were produced as described in Examples 1-4. The vascular surgeonmade 40 intramuscular injections of 1 mL aliquots of cells or stem cellsderived from umbilical cord into previously identified locations alongthe ischemic limb using ultrasound guidance. Procedures were carried outunder local anesthesia and conscious sedation.

Patients were evaluated at 1, 4, 8, 12 and 26 weeks post-procedure.Clinical outcomes included amputation status, Rutherford classificationof limb ischemia, and pain as determined by Visual Analog Scale (VAS).Major amputations were defined as those occurring above the ankle.Hemodynamic outcome was evaluated by Ankle Brachial Index (ABI).Laboratory monitoring of hematology and blood chemistries was alsoperformed. Ophthalmologic retinal examination was performed at baselineand 12 weeks in diabetics to evaluate for proliferative retinopathy.Results are shown in FIGS. 11A and 11B. Injection only represents thedelivery of stem cells, while the control was a saline solution lackingthe stem cells.

Example 6—Therapeutic Use of Cells or Stem Cells from Umbilical Cord forTreating Chronic Refractory Angina and/or Congestive Heart Failure

Patients with Canadian Cardiovascular Society (CCS) class III-IV anginadespite maximal medical or surgical therapy who were ineligible forfurther percutaneous or surgical revascularization (based on coronaryanatomy) and who had evidence for reversible ischemia on an exercisesingle photon emission computed tomography (SPECT) were enrolled.

Cells were produced as described in Examples 1-4. The femoral vein wascannulated with a 7 French sheath, a 6 French catheter was placed in thecoronary sinus and a 0.035 mm hydrophilic guide wire was placed in theinterventricular or lateral vein followed by placement of a peripheralballoon into the mid portion of the coronary sinus to allow nonselectivedelivery of cells. (Cook Medical, Indiana, USA). The balloon wasinflated at very low pressure (1 to 2 atm) for 10 minutes producingstagnation of the flow. 50 mls of cells (50,000,000-400,000,000) wereinjected manually through the balloon at a rate of 10 mls per minute.The average total procedure time for cell delivery was 30 minutes. FIG.12 shows an angiogram demonstrating delivery of cells into the heartusing a retrograde technique.

The baseline screening assessment of patients included clinicalevaluation, electrocardiogram (ECG), laboratory evaluation (completeblood count, blood chemistry, erythrocyte sedimentation rate, creatinekinase, and troponin T serum levels). Patients kept a record of dailyangina frequency for three weeks, and the severity of angina was gradedaccording to the CCS class at baseline, 3, 12, and 24 months. Within twoweeks prior to cell therapy, exercise capacity was evaluated usingbicycle ergometry in conjunction with SPECT imaging to assess myocardialischemia and left ventricular (LV) function.

Example 7—Heart Failure Safety Study

Ten patients, 5 ischemic and 5 non-ischemic, received retrogradedelivery of cells to the heart as described in Example 6. FIGS. 13A-Dshows time lapse images of such a retrograde delivery. The baselinescreening assessment of patients included clinical evaluation,electrocardiogram (ECG), laboratory evaluation (complete blood count,blood chemistry, erythrocyte sedimentation rate, creatine kinase, andtroponin T serum levels). Patients were given follow up assessments at1, 3, 6, and 12 months. Tables 1 and 2 show results over time forischemic and non-ischemic patients.

TABLE 1 Ischemic Baseline 1 month 3 month Troponin 0.03 0.02 0.02 BNP543 320 178 EF % 26 33 38 6 m.w. 255 260 344 VO₂Max 14 15 17 AE/SAE 0/01/0 1/0

TABLE 2 Non-Ischemic Baseline 1 month 3 month Troponin 0.03 0.03 0.02BNP 655 389 156 EF % 22 34 39 6 m.w. 227 235 312 VO₂Max 13 15 19 AE/SAE0/0 0/0 1/0

Example 8—Therapeutic Use of Cells or Stem Cells from Umbilical Cord forDiabetes

Cells are produced as described in Examples 1-45. Therapeutic doses canbe 50,000,000-400,000,000. The cells are delivered thru arterial accessinto the celiac and or SMA artery, thereby delivering cells into thehead and/or tail of the pancreas via infusion technique.

Example 9—Therapeutic Use of Cells or Stem Cells from Umbilical Cord forTreating COPD/Asthma/Emphysema

The following inclusion criteria were used for subjects in this study.Individuals were included having:

-   -   moderate or severe COPD with a post-bronchodilator FEV1/FVC        ratio <0.7    -   subject must have a post-bronchodilator FEV1% predicted value        ≥30%    -   current or ex-smoker, with a cigarette smoking history of >20        pack-years

Subjects exhibiting one or more of the following were excluded from thestudy:

-   -   diagnosed with asthma or other clinically relevant lung disease        other than COPD (e.g. restrictive lung diseases, sarcoidosis,        tuberculosis, idiopathic pulmonary fibrosis, bronchiectasis, or        lung cancer)    -   diagnosed with α1-Antitrypsin deficiency    -   body mass greater than 150 kg or less than 40 kg    -   subject has an active infection    -   subject has had a significant exacerbation of COPD or has        required mechanical ventilation within 4 weeks of screening    -   uncontrolled heart failure, atrial fibrillation    -   cardiopulmonary rehabilitation initiated within 3 months of        screening    -   subject has evidence of active malignancy, or prior history of        active malignancy that has not been in remission for at least 5        years    -   subject has a life expectancy of <6 months

Cells are produced as described in Examples 1-4. Therapeutic doses canbe 50,000,000-400,000,000 cells. While a subject is sitting upright thecells are administered simultaneously thru an aerosolized delivery whichwill remain top half of the lung due to normal physiologic ventilationperfusion and is given intravenous which is delivered to the lower halfof the lung, due to the natural ventilation perfusion for a personsitting upright. This combined technique is used due to the fact thateither one performed alone does not deliver sufficient biologic to theentire lung volume.

20 test subjects were divided into 4 groups and received the following:

5 subjects in Group 1 were given placebo—saline injection

5 subjects in Group 2 were given IV delivery—200M cells

5 subjects in Group 3 were given inhaled delivery—200M cells

5 subjects in Group 4 were given IV and inhaled delivery—100M/100M cells

Results obtained from these groups treated with no cells, IV only,inhaled only and both IV and inhaled are shown in Table 3.

Regarding aersolization, cells were prepared as described, suspended in1-5 mls of saline and aerosolized at a pressure of 30 psi for 8-10minutes

TABLE 3 IV and Placebo IV Inhaled Inhaled Group 1 Group 2 Group 3 Group4 FEV1/FVC 0.55 ± 0.15 0.49 ± 0.08 0.51 ± 0.10 0.47 ± 0.07 pre FEV1/FVC0.52 ± 0.13 0.53 ± 0.12 0.57 ± 0.11 0.66 ± 0.05 post O₂ L/min pre 3.0 ±1.0 2.8 ± 1.2 3.2 ± 1.0 2.8 ± 1.2 O₂ L/min post 3.2 ± 1.2 2.4 ± 1.4 2.5± 1.2 2.0 ± 1.0 MAP/CE 2 0 0 0

Example 10—Therapeutic Use of Cells or Stem Cells from Umbilical Cordfor Treating Wound Healing

Cells are produced as described in Examples 1-4. Therapeutic doses canbe 50,000,000-400,000,000 cells in this example. Cells are delivered tothe wound via injection and/or aerosolized in a PL-carrier with additionof liquid calcium and thrombin.

Example 11—Therapeutic Use of Cells or Stem Cells from Umbilical Cordfor Orthopedic Applications

Cells are produced as described in Examples 1-4. Therapeutic doses canbe 50,000,000-400,000,000 cells in this example. Under ultrasoundguidance the cells are directly injected into the intraarticularspace/joint with or without a microfracture technique. They cells mayalso be delivered with PRPL or PL carrier in addition to liquidcalcium/thrombin. As one example, FIGS. 14A and 14B show images of theknee of an 80 year old female prior to the delivery procedure. FIGS. 14Cand 14D show images of the same knee from the same 80 year old female 3months post-transplant. It is noted that more intraarticular space isobserved in the patient in the post-transplant images.

Example 12—Therapeutic Use of Cells or Stem Cells from Umbilical Cordfor Acute Radiation Syndrome Applications in Mice

Female C57BL/6J mice were used as the recipient population. Umbilicalcord stem cells were isolated as previously described but isolated inthis case from mice. The female C57BL/6J mice received TBI using aCs-137 radiation source. Lethal irradiation was performed using 9.5 Gy.Within 8 hours post irradiation mice received transplants intravenously.Evaluation of peripheral blood counts of animals treated with stem cellsrevealed similar leukocyte and thrombocyte recovery as observed inrecipients treated with HSCs. (See FIGS. 15A-B) Seven months posttransplantation recipients were hematologically well with a normaldistribution of peripheral blood cell populations. (See Table 4).

TABLE 4 Peripheral blood cell population in transplanted micelymphocytes neutrophils monocytes eosinophils 72% +/− 3 21% +/− 3 5% +/−2 2% +/−1

Example 13—Therapeutic Use of Cells or Stem Cells from Umbilical Cordfor Acute Radiation Syndrome Applications in Humans

In order to determine if human derived subepithelial layer umbilicalcord cells had the same effect as Example 12, the same experiment wasrepeated using human-derived cells as the donor material and nod/scidgamma(c) null mice as the recipient. Animals were treated as previouslydescribed and transplanted IV at 6, 12 and 24 hours post total bodyirradiation (TBI). 6 months post transplant all (n=30) control mice thatdidn't receive cells post TBI were dead. FIG. 16 shows the survival ofmice receiving human cells 6, 12 and 24 hours post TBI.

Of course, it is to be understood that the above-described arrangementsare only illustrative of the application of the principles of thepresent disclosure. Numerous modifications and alternative arrangementsmay be devised by those skilled in the art without departing from thespirit and scope of the present disclosure and the appended claims areintended to cover such modifications and arrangements. Thus, while thepresent disclosure has been described above with particularity anddetail in connection with what is presently deemed to be the mostpractical embodiments of the disclosure, it will be apparent to those ofordinary skill in the art that numerous modifications, including, butnot limited to, variations in size, materials, shape, form, function andmanner of operation, assembly and use may be made without departing fromthe principles and concepts set forth herein.

What is claimed is:
 1. A culture of an isolated cell prepared by aprocess comprising: placing a subepithelial layer of a mammalianumbilical cord tissue in direct contact with a growth substrate; andculturing the subepithelial layer such that the isolated cell from thesubepithelial layer is capable of self-renewal and culture expansion,wherein culturing comprises culturing in a culture media that is free ofanimal components, wherein the isolated cell expresses at least threecell markers selected from the group consisting of CD29, CD73, CD90,CD166, SSEA4, CD9, CD44, CD146, or CD105, and wherein the isolated celldoes not express NANOG and at least five cell markers selected from thegroup consisting of CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19,CD117, Stro-1, or HLA-DR.
 2. The culture of claim 1, wherein theisolated cell expresses CD29, CD73, CD90, CD166, SSEA4, CD9, CD44,CD146, and CD105.
 3. The culture of claim 1, wherein the isolated celldoes not express CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19, CD117,Stro-1, and HLA-DR.
 4. The culture of claim 1, wherein the isolated cellis positive for SOX2.
 5. The culture of claim 1, wherein the isolatedcell is positive for OCT4.
 6. The culture of claim 1, wherein theisolated cell is positive for SOX2 and OCT4.
 7. The culture of claim 1,wherein the wherein the isolated cell is capable of differentiation intoa cell type selected from the group consisting of adipocytes,chondrocytes, osteocytes, cardiomyocytes, endothelial cells, andmyocytes.
 8. The culture of claim 1, wherein the isolated cell producesexosomes expressing CD63, CD9, or CD63 and CD9.
 9. The culture of claim1, wherein placing and culturing the subepithelial layer is performedwithout the use of any enzyme.
 10. A culture of differentiated cellsderived from the culture of the isolated cell of claim 1, wherein theculture of differentiated cells includes a cell type selected from thegroup consisting of adipocytes, chondrocytes, osteocytes,cardiomyocytes, endothelial cells, myocytes and combinations thereof.11. The culture of claim 1 that has been differentiated into a cultureof adipocyte cells.
 12. The culture of claim 1 that has beendifferentiated into a culture of chondrocyte cells.
 13. The isolatedcell of claim 1 that has been differentiated into a culture of osteocytecells.
 14. The culture of claim 1 that has been differentiated into aculture of cardiomyocyte cells.
 15. The culture of claim 1 that has beendifferentiated into a culture of myocyte cells.